【作者】 张纲；

【导师】 李春岩；

【作者基本信息】 河北医科大学 ， 神经病学， 2006， 博士

【摘要】 格林巴利综合征(GBS)是目前世界上由自身免疫性神经肌肉疾病导致软瘫首要的病因。神经节苷脂，一种携带唾液酸基的鞘糖脂，在脊椎动物的神经系统中含量丰富。拮抗神经节苷脂的抗体与多种神经系统疾病有关目前，有充足的证据证明抗神经节苷脂抗体与某些GBS的亚型有重要的关联。但是，仍有很多与神经节苷脂抗体在此类疾病中作用有关的重要议题有待证明。诸如，有关神经节苷脂抗体的目标抗原本质问题，提供进一步的与补体作用有关的实验证据等。充分理解上述有关神经节苷脂抗体诱导损伤的分子和细胞水平的作用机制可以帮助我们更有效地开发相关的免疫疗法。为达到这一目的，一个很重要的步骤就是建立一种可模拟此类损伤的体外模型。这是由于目前我们还缺乏一种稳定而显著的小鼠GBS动物模型。因此，我们建立了两种相关的体外模型：(1)抗神经节苷脂抗体诱导的神经细胞毒模型，(2)一种由神经节苷脂抗体导致了运动神经损伤的脊髓培养模型。另外，正是由于目前缺乏一种行之有效的GBS被动免疫模型，因此有关神经节苷脂抗体在GBS中所起作用还有争议。我们最近培育出一些具有相对高亲和力的单克隆抗神经节苷脂抗体。我们尝试了通过腹腔移植分泌这些抗体的杂交瘤的方法，将抗体被动转移至小鼠体内。而后，我们通过此模型研究了诸多在神经病变产生过程中可能有关联的因素。虽然IVIg已通过临床研究被证明为一种有效的治疗急慢性免疫介导的神经病变(包括格林巴利综合征)的一线疗法。但是，目前还不曾系统地检验过不同品种，或同一品种但不同批次的IVIg，是否具有相同或相似的治疗效果。为了验证这一议题，我们利用了一种体外神经节苷脂抗体诱导的细胞毒模型，分析和比较了8种品牌的IVIg，以及来自其中两种品牌的不同批次IVIg在此模型中的保护作用。第一部分：神经节苷脂抗体诱导的神经细胞细胞毒模型，及IVIg保护作用的相关研究目的：探讨两类与神经节苷脂抗体所导致的神经损伤相关的基本问题：(1)有些神经节苷脂抗体与糖蛋白有交叉反应，因此此类抗体的目标抗原尚待进一步阐明。(2)虽然，目前病理学研究已提示补体激活在GBS发病中的作用，但依旧缺乏这方面的实验学依据。方法：一种建立在NG108-15和NG-CR-72细胞上的由不同抗神经节苷脂单克隆抗体或含有抗神经节苷脂抗体的GBS患者血清诱导的神经细胞细胞毒模型被在此研究中使用。另外，人静脉免疫球蛋白(Human Intravenous Immunoglobulin，IVIg)也被用于此模型来检验其可能存在的保护作用。结果：(1)无论是抗神经节苷脂单克隆抗体，还是含有神经节苷脂抗体的GBS病人血清，均可通过细胞表面相应的特异神经节苷脂而导致神经细胞溶解；(2)这类细胞溶解还是补体依赖性的。此外，在本实验模型中，GD1a的膜聚集区较GM1的聚集区对特异性神经节苷脂抗体诱导的损伤具有更高的敏感性。(3)IVIg可有效地抑制此模型中由神经节苷脂抗体诱导的细胞毒作用。IVIg在此模型中的保护作用机制可归结于：抗个体基因型抗体作用，以及其对补体激活的抑制作用。结论：此实验模型因此可被应用于鉴别由免疫介导的神经疾病患者体内的致病神经节苷酯抗体，同时还可被用来筛选新的或实验性的用于防治神经节苷酯抗体诱导神经损伤的治疗方法。第二部分：神经节苷脂抗体分泌型杂交瘤导致的小鼠神经病变目的：目前由于缺乏被动免疫动物模型，对于神经节苷脂抗体在GBS发病中所起的作用仍存在争议。因此，本研究具体探讨了可影响神经节苷脂抗体对神经损伤的一些因素，并希望进而对以后成功开发出被动免疫动物模型起到抛砖引玉的作用。方法：我们在以往的研究中培养出多种单克隆神经节苷脂抗体。某些此类抗体被通过腹腔神经节苷脂抗体分泌型杂交瘤移植术，或系统性全身施药法而被动转移到小鼠体内。实验结束后，上述动物接受了系统地病理学检验，以观察其神经损伤状况。同时，比较了两组间血-神经屏障通透性的差异。结果：大约一半接受腹腔杂交瘤移植术的小鼠出现了影响到部分神经纤维的，散在的轴索病变。与之形成对比的是，被动系统性地接受抗神经节苷脂抗体的动物，尽管血循环中抗体滴度很高，但并未发生神经纤维退变。通过对血-神经屏障通透性的研究发现，接受腹腔杂交瘤移植术的动物有着较高的血-神经屏障通透性。结论：我们的实验结果表明神经病变的形成除了需要循环中的致病抗体外，其他诸如，抗体的可达性及神经纤维对抗体介导损伤的耐受性等因素均可发挥作用。第三部分：一种具有高亲和力单克隆抗体导致的体外运动神经退行性病变目的：评估神经节苷脂抗体对运动神经损伤的作用。方法：近期我们培养出两种含有高亲和性的抗GD1a的单克隆抗体，这两种抗体对大鼠脊髓体外培养中运动神经的损伤被在此研究中得以评估。结果：一种具有补体激活能力的抗GD1a单克隆抗体不仅导致脊髓体外培养中运动神经元数目的明显下降，并引起运动神经纤维Wallerian变性。与此同时，抗体亚型匹配的对照抗体及拮抗GD1a的非补体激活性抗体均不能造成明显的运动神经病变。结论：本研究证实此体外脊髓培养模型对研究运动神经元及神经纤维的免疫性损伤是非常有意义的。抗神经节苷脂抗体对运动神经体外损伤的发现有力地支持了神经节苷脂抗体在GBS运动轴索亚型发病过程中起到重要作用的这一理念。第四部分：多种IVIg在一种体外免疫神经疾病模型中的评估目的：分析和比较不同品牌及不同批号的静脉免疫球蛋白IVIg的保护作用。方法：我们利用了一种免疫神经疾病的细胞模型，由神经节苷脂抗体诱导的神经细胞(NG108-15)细胞毒模型，分析比较了八种经FDA批准的可在美国使用的不同品牌，以及其中两种品牌不同批号的IVIg在此细胞毒模型中的保护功效。结果：这些不同品牌或不同批号的产品具有非常相似的对此模型中由神经节苷脂抗体引导细胞损伤的保护作用。结论：这说明不同的IVIg在格式化模型中所展示的作用是具有可比性的。更多还原

【Abstract】 Guillain-Barrésyndromes (GBS) currently are the world’s leading causeof acute autoimmune neuromuscular paralysis. Gangliosides, sialicacid-bearing glycosphingolipids, are highly enriched in the vertebrate nervoussystem. Anti-ganglioside antibodies are associated with various humanneuropathies. Different lines of investigation have found strong evidence ofthe pathogenic role of anti-ganglioside antibodies in some forms of GBS.However, there’re still plenty of important issues related with the role ofanti-ganglioside antibody remain unproven. For example, the nature ofantigens targeted by anti-ganglioside Abs has not been fully addressed, whichis crucial to any autoimmune disease; the experimental data for the role ofcomplement also remain inconclusive, etc. Understanding those cellular andmolecular mechanisms involved in the pathogenesis of anti-gangliosideantibody mediated injury is crucial to the development of specificimmunotherapies. A critical step in this regard is the generation of in vitromodels of such injury. Currently, lack of a robust mouse model of GBSprecludes addressing this question in vivo. To address these issues wedeveloped two ex-vitro models: (ⅰ) anti-ganglioside Ab-mediated neuronalcytotoxicity assay; (2) Organotypic spinal cord cultures system, in whichanti-ganglioside antibody caused motor nerves degeneration.Also, because of lack of a passive transfer model, the role ofanti-ganglioside antibodies in GBS continues to be debated. We recently haveraised several high-affinity monoclonal IgG anti-ganglioside antibodies. Inthis study, we try passively transfer these antibodies by intraperitonealhybridoma implantation in mice. Then, we studied the possible factors whichcould play role in the development of neuropathy.Finally, in controlled clinical studies, intravenous immunoglobulin (IVIg) has been shown to be effective as first-line therapy for both acute and chronicimmune neuropathies that include Guillain-Barre’ syndrome (GBS). Whetheror not, the efficacy of different IVIg brands, or different lots of the same brand,can affect the beneficial response differently in patients remain unclear. And, ithas never been systematically examined before. To address this question, weexamined the protective efficacy of different brands of IVIg and different lotsof the two brands in the in vitro anti-ganglioside Ab-mediated neuronalcytotoxicity model.PartⅠ: Anti-ganglioside antibody-mediated neuronal cytotoxicity and itsprotection by intravenous immunoglobulin: implications for immuneneuropathiesPurpose:Study two basic issues related to antigangliosideantibody-mediated neural injury: (1) some anti-ganglioside antibodies cancross-react with glycoproteins and therefore the nature of antigens targeted bythese antibodies is not well established; and (2) although pathological studiessuggest that complement activation occurs in GBS, experimental data for therole of complement remain inconclusive.Method: we developed and characterized a simple anti-gangliosideantibody mediated cytotoxicity assay, which is based on two neuronal cell line:NG108-15 and it’s mutant cell line, NG-CR-72 cell. In this assay, weadministrate both anti-gangliosides monoclonal antibodies and GBS patients’sera having anti-ganglioside activity. IVIg’s (human intravenousimmunoglobulin) protection effect was also being tested in this model.Results: Our results demonstrate first, that both GBS sera containinganti-ganglioside antibodies and monoclonal anti-ganglioside antibodies causeneuronal cell lysis by targeting specific cell surface gangliosides, and secondly,that this cell lysis is complement dependent. In this assay, the GD1a cellmembrane pool appears to be more susceptible to antigangliosideantibody-mediated injury than the GM1 pool. Further, IVIg significantlydecreased cytotoxicity in this assay. Our data indicate that the mechanisms ofIVIg-mediated protection in this assay include anti-idiotypic antibodies, and down-regulation of complement activation.Conclusion: This simple cytotoxicity assay can potentially be used forscreening of (ⅰ) pathogenic anti-ganglioside antibodies in patients withimmune-mediated neuropathies; and (ⅱ) new/experimental therapies toprevent anti-ganglioside antibody-mediated neural injury.PartⅡ: An Anti-ganglioside Antibody-Secreting Hybridoma InducesNeuropathy in MicePurpose: Currently, the role of anti-ganglioside antibodies in GBScontinues to be debated because of lack of a passive transfer model. Severalpossible factors which could affect the role of anti-ganglioside antibody innerve injury were discussed in this study. We hope it can help developing thesuccessful in-vivo animal passive transfer model.Methods:We recently have raised several monoclonal IgGanti-ganglioside antibodies. We passively transfer these antibodies byintraperitoneal hybridoma implantation and by systemic administration ofpurified anti-ganglioside antibodies in mice. Finally, the pathological studywas done on those animals, and the nerve injury was evaluated. Theblood-nerve barrier in these animals was also being studied.Results:Approximately half the animals implanted with anintraperitoneal clone of anti-ganglioside antibody-secreting hybridomadeveloped a patchy, predominantly axonal neuropathy affecting a smallproportion of nerve fibers. In contrast to hybridoma implantation, passivetransfer with systemically administered anti-ganglioside antibodies did notcause nerve fiber degeneration despite high titre circulating antibodies.Blood-nerve barrier studies indicate that animals implanted with hybridomahad leaky blood-nerve barrier compared to mice that received systemicallyadministered anti-ganglioside antibodies.Conclusion: Our findings suggest that in addition to circulatingantibodies, factors such as antibody accessibility and nerve fiber resistance toantibody-mediated injury play a role in the development of neuropathy.PartⅢ: A high-affinity monoclonal anti-ganglioside antibody causes degeneration of motor nerves in vitroPurpose: To study the anti-ganglioside’s role in the motor nerve injury.Methods: Recently we generated two high affinity IgG anti-GD1amonoclonal antibodies whose toxic effect on motor nerves in organotypic ratspinal cord cultures was studied.Results: Organotypic spinal cord cultures incubated with acomplement-fixing monoclonal anti-GD1a-related antibody demonstrated asignificant drop in motor neuron numbers and showed evidence ofWallerian-like degeneration of nerves. Isotype-matched sham antibody andnon-complement-fixing anti-GD1a antibody did not induce significantdegeneration of motor nerves.Conclusion: Our results indicate that the spinal cord culture system is auseful in vitro model for studying immune injury to motor neurons and nerves.The observation of in vitro cytotoxicity of anti-ganglioside antibody to motornerves supports the pathogenic role of anti-ganglioside antibodies found in theAMAN variant of GBS.PartⅣ: Comparison of different brands of IVIg in an in vitro model ofimmune neuropathyPurpose: To analyze and compare the protection efficacy of differentbrands or different lots of Intravenous immunoglobulin’s (IVIg).Methods: we compared the efficacy of eight FDA approved brandsand/or lots of two brands of IVIg in a cell culture model of immuneneuropathy.Results: We report that products examined were equally effective andthere was no lot-to-lot variability in our experimental model.Conclusion: These findings support the notion that efficacy of differentIVIg products is comparable in a standardized model.更多还原